Biocompatible phase invertible proteinaceous compositions and methods for making and using the same

ABSTRACT

Biocompatible phase invertible proteinaceous compositions and methods for making and using the same are provided. Phase invertible compositions in accordance with the invention are prepared by combining a liquid proteinaceous substrate and a liquid crosslinking composition, where the liquid crosslinking composition includes a macromolecular crosslinking agent. Also provided are kits for use in preparing the subject compositions. The subject compositions, kits and systems find use in a variety of different applications.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/326,835, filed Dec. 2, 2008, which claims priority to U.S.Provisional Patent Application No. 60/991,867, filed Dec. 3, 2007; thefull disclosures of which application are herein incorporated byreference.

INTRODUCTION

A number of sealant compositions have become available to control fluidleakage at a surgical site, as well as for other applications. However,currently available sealant compositions may suffer from seriouslimitations with regards to the field in which they can be used, as wellas their biocompatibility and their physical properties. Side effects,such as inflammation, acute fibrous formation at the wound site,toxicity, inability to be used in a bloody field, poor physicalproperties of the sealant, and poor adhesion to the surgical site, mayhave a serious impact on the patient and resultantly may play asignificant role in the long term efficacy of the repair. Further,useful sealants have properties that can render them more effective forsurgical application. Characteristics, such as the ability to belocalized to a specific location, adequately long or shortpolymerization times, and adequate in vivo resorption characteristics,are vital to a successful completion of the sealing procedure.

As such, there is a continued need for the development of newbiocompatible compositions for use as sealants, as well as for use inother applications.

SUMMARY

Biocompatible phase invertible proteinaceous compositions and methodsfor making and using the same are provided. Phase invertiblecompositions in accordance with aspects of the invention are prepared bycombining a liquid proteinaceous substrate and a liquid crosslinkingcomponent, where the liquid crosslinking component includes amacromolecular crosslinking agent.

The macromolecular crosslinking agent is produced by combining an excessof a crosslinking agent, e.g., a heat-treated dialdehyde, with an amountof a physiologically acceptable polymer, such as a glycosaminoglycan.The excess of crosslinking agent and physiologically acceptable polymerreact to produce a macromolecular crosslinking agent. Upon combinationof the macromolecular crosslinking agent with the proteinaceoussubstrate, the macromolecular crosslinking agent reacts with proteins inthe substrate component to produce a final composition characterized bythe presence of an interpenetrating network.

Also provided are kits for use in preparing the subject compositions.The subject compositions, kits and systems find use in a variety ofdifferent applications.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 provides a table of results observed with different sealantcompositions, as described in the Experimental Section, below.

DETAILED DESCRIPTION

Biocompatible phase invertible proteinaceous compositions and methodsfor making and using the same are provided. The subject phase invertiblecompositions are prepared by combining a liquid proteinaceous substrateand a liquid crosslinking composition, where the liquid crosslinkingcomposition includes a macromolecular crosslinking agent. Also providedare kits for use in preparing the subject compositions. The subjectcompositions, kits and systems find use in a variety of differentapplications.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing the subject invention, phase invertiblecompositions in accordance with the invention are described first ingreater detail, followed by a review of applications in which thecompositions find use, as well as a review of kits and systems that finduse in making or using the subject phase invertible compositions.

Biocompatible Phase Invertible Proteinaceous Composition

As summarized above, the subject invention provides a biocompatiblephase invertible proteinaceous composition. The composition is one that,over time, undergoes a phase inversion from a first, liquid state to asecond, solid state. In the first, liquid state, the phase invertiblecomposition has a viscosity sufficient such that it can be caused toflow through a medical canula or sprayed onto a target tissue site. Thephase invertible compositions are characterized by being capable ofbonding tissue in both wet (e.g., blood) and dry environments, whereadhesion of the composition to the tissue is exceptionally strong. Anaspect of the compositions is that, once they are applied to a targettissue location, they remain at the target tissue location. A furtheraspect of the subject compositions is that they are well-tolerated bythe body and do not elicit a substantial, if any, inflammatory response.

The subject phase invertible proteinaceous compositions are prepared bycombining or mixing a liquid proteinaceous substrate with a liquidcrosslinking composition. Each of these precursor components orcompositions is now reviewed separately in greater detail.

Crosslinker Composition

As indicated above, the phase invertible composition is produced bycombining a liquid proteinaceous substrate, as described above, with aliquid crosslinker composition that includes a macromolecularcrosslinking agent. While the viscosity of the crosslinking componentmay vary, in certain embodiments it approximates the viscosity of theproteinaceous component, expressed in centistokes (cSt) at about25.degree. C., ranging from 10 cSt to 150 cSt, such as 30 cSt to 70 cSt.The crosslinking component may be sterilized according to any convenientprotocol, where sterilization protocols of interest include, but are notlimited to: gamma, electron beam, and the like. The liquid crosslinkingcomponent is one that is storage stable. By storage stable is meant thatthe substrate may be maintained under storage conditions, such as roomtemperature for a period of time of at least 1 years or longer, such as3 years or longer, without undergoing any substantial change thatnegatively impacts the function of the substrate such that it is nolonger suitable for use in preparing a biocompatible phase invertiblecomposition of the invention.

The crosslinking component is one that is produced by combining anexcess of a crosslinking agent with an amount of a physiologicallyacceptable polymer. Examples of crosslinking agents include, but are notlimited to: photo-oxidative molecules; carbodimides; carbonyl containingcompounds, e.g., mono- and dicarbonyls, including carboxilic acids,e.g., dicarboxylic acids, such as adipic acid, glutaric acid and thelike, and aldehydes, including mono- and dialdehydes, e.g.glutaraldehyde; etc. In certain embodiments, the crosslinker employed isan aldehyde crosslinker. In certain of these embodiments, the aldehydecrosslinker is pretreated to produce a stabilized aldehyde crosslinker,for example, a stabilized glutarhaldehyde crosslinker, e.g., where thecrosslinker is heat stabilized aldehyde, such as heat stabilizedglutaraldehyde (such as described in U.S. Pat. No. 7,303,757, thedisclosure of which is herein incorporated by reference).

While the amount of crosslinking agent in the composition may vary, incertain embodiments the amount of crosslinking agent ranges from 0.1 to20% (v/v), such as 0.5 to 15% (v/v) and including 1 to 10% (v/v).

In addition to the cross-linking agent, the crosslinking compositionfurther includes an amount of a physiologically acceptable polymer. Thephysiologically acceptable polymer may be any polymer that is toleratedby the body and reacts with the crosslinking agent to produce aprepolymer macromolecular crosslinking product. The prepolymer productis one that is a reaction product between the crosslinking agent and thepolymer, and includes a polymer backbone with one or more crosslinkingmolecules covalently bonded thereto such that the polymer backboneincludes one or more crosslinking functional groups, where thecrosslinking functional groups include at least one reactive moiety,e.g., an aldehyde moiety, that can covalently bond to the proteincomponent of the proteinaceous substrate. As such, the prepolymerproduct retains the ability to crosslink upon contact with theproteinaceous substrate. The prepolymer product of certain embodimentsis a soluble macromolecule that comprises a polymeric backbone moleculebound to crosslinking agent molecules, where at least a portion of thebound crosslinking agent molecules retain a free crosslinking moietythat can bind proteins in the proteinaceous substrate. This prepolymerproduct “macromolecular” crosslinking agent may vary in averagemolecular weight, and in certain embodiments may range in weight from10,000 to 4 million Daltons, such as 500,000 to 2 million Daltons.

In certain embodiments, the physiologically acceptable polymer is aglycosaminoglycan (i.e., a mucopolysaccharide). Specificglycosaminoglycans of interest include, but are not limited to:chondroitin sulphate; dermatan sulphate; keratan sulphate; heparin;heparan sulphate; and hyaluronan (i.e., hyaluronic acid). In certainembodiments, the glycosaminoglycan component is hyaluronan.

The crosslinking agent is present in the crosslinking composition inexcess with respect to the amount of physiologically acceptable polymer.In certain embodiments, the amount of physiologically acceptable polymermakes up from 0.01 to 5% (w/v), such as 0.05 to 3% (w/v) including 0.1to 1% (w/v) of the crosslinking composition.

In certain embodiments, the crosslinker composition may further includean amount of a viscosity modifying agent. Viscosity modifying agents ofinterest include, but are not limited to: polyoxyethylene orpolyoxypropylene polymers or copolymers thereof, such as polyethyleneglycol and polypropylene glycol; nonionic cellulose ethers such asmethylcellulose, ethylcellulose, hydroxymethylcellulose,hydroxyethylcellulose, carboxymethylcellulose, carboxyethylcellulose andhydroxypropylcellulose; additional celluloses, such ascarboxymethylcellulose sodium, carboxymethylcellulose calcium,carboxymethylstarch; and the like. In certain embodiments of particularinterest, the emulsifying agent is a cellulose ether, particularly anonionic cellulose ether, such as carboxymethylcellulose.Carboxymethylcellulose is available from a variety of commercialsources, including but limited to, Sigma, Hercules, Fluka and Noviant.In certain embodiments, the average molecular weight of the celluloseether is at least about 1000 Daltons, such as at least about 5000Daltons, where the average molecular weight may be as high as 10,000Daltons or higher, e.g., 50,000 Daltons or higher, 100,000 Daltons orhigher, and ranges in certain embodiments from about 5,000 to about100,000 Daltons, such as from about 10,000 to about 50,000 Daltons. Theproportion of the viscosity modifying agent in the sealant in certainembodiments ranges from 0.01 to 10% (v/v), such as 0.1 to 4% (v/v)including 0.5 to 2% (v/v).

Proteinaceous Substrate

The liquid proteinaceous substrate from which the subject phaseinvertible compositions are prepared is a liquid composition, e.g., anaqueous composition, that is made up of at least a proteinaceouscomponent and, in certain embodiments, an adhesion modifier, where thesubstrate may include one or more additional components, including, butnot limited to: a plasticizer; a carbohydrate; and the like.

The substrate may be sterilized according to any convenient protocol,where sterilization protocols of interest include, but are not limitedto: gamma radiation, electron beam and the like.

The liquid proteinaceous substrate is one that is storage stable. Bystorage stable is meant that the substrate may be maintained understorage conditions, such as room temperature for a period of time of atleast 1 years or longer, such as 3 years or longer, without undergoingany substantial change that negatively impacts the function of thesubstrate such that it is no longer suitable for use in preparing abiocompatible phase invertible composition of the invention.

Proteinaceous Component

The proteinaceous component (i.e., proteinaceous material) of thesubstrate is made up of one or more distinct proteins. The proteins ofthis component may be either synthetic or naturally occurring proteins,where the proteins may be obtained/prepared using any convenientprotocol, e.g., purification from naturally occurring sources,recombinant production, synthetic production, and the like, where incertain embodiments the proteins are obtained from naturally occurring,e.g., bovine, porcine or human, sources. Specific proteins of interestinclude, but are not limited to: albumins, collagens, elastins, fibrins,and the like.

The amount of protein in the substrate composition may vary, where thespecific selection of concentration is dependent on the desiredapplication and product parameters desired therefore, such as tenacity,hardness, elasticity, resorption characteristics and plateletaggregation effects. In certain embodiments, the total protein totalconcentration in the substrate compositions ranges from about 1 to 75%(w/w), such as 1-50% (w/w), including 5 to 40% (w/w).

In certain embodiments, the primary protein of the substrate compositionof this embodiment is albumin, where the albumin may be a naturallyoccurring albumin, e.g., human albumin, bovine albumin, porcine albuminetc., or a variant thereof. As is known in the art, the albumin may bepurchased in powdered form and then solubilized into an aqueoussuspension, or alternately, may be purchased in aqueous form. Purifiedalbumin may derived from any one of a number of different sourcesincluding, bovine, ovine, equine, human, or avian in accordance to wellknown methods (ref.: Cohn et. Al, J. Amer. Chem. Soc. 69:1753) or may bepurchased in purified form from a supplier, such as Aldrich Chemical(St. Louis, Mo.), in lyophilized or aqueous form. The albumin may bederivatized to act as a carrier for drugs, such as heparin sulfate,growth factors, antibiotics, or may be modified in an effort to moderateviscosity, or hydrophilicity. Derivatization using acetylating agents,such as, but not limited to, succinic anhydride, and lauryl chlorides,are useful for the production of binding sites for the addition ofuseful molecules. In these embodiments where the proteinaceous componentincludes albumin, the albumin may be present in concentrations rangingfrom about 10 to about 50% (w/w), such as from about 30 to about 40%(w/w).

In certain embodiments, the proteinaceous component also includes acollagen, e.g., a naturally occurring collagen (human, bovine, porcine)or synthetic variant thereof. In accordance with the invention, thecollagen may be in dry or aqueous forms when mixed with the albumin.Collagen may be derivatized to increase it utility. Acylating agents,such as anhydrides or acid chlorides, have been found to produce usefulsites for binding of molecules such as growth factors, and antibiotics.When present, the collagen sometimes ranges from about 1 to about 20%(w/w), including from about 1 to about 10% (w/w), such as from about 1to about 4% (w/w), including from about 2 to 4% (w/w).

The subject proteinaceous component, as described above, may or may notinclude one or more active agents, e.g., drugs, present in it, asdesired. When present, the agent(s) may be bound to the polymers, asdesired.

Tackifying Agent

Also present may be one or more tackifying agents. Tackifying agentsimprove the adhesiveness of the sealant to the biological surface. Inmany embodiments, the adhesion modifiers are polymeric compounds havingcharged functionalities, e.g., amines, etc. Whereas numerous tackifyingagents may be used, one of particular applicability is polyethyleneimine(PEI). PEI is a long chain branched, alkyl polymer containing primary,secondary and tertiary amines. The presence of these highly ionic groupsresults in significant attachment through ionic interactions with theunderlying surface. In addition, the presence of PEI in the substratesignificantly enhances the presence of amine terminals suitable toproduce crosslinks with the crosslinking agent. Additional tackingagents of interest include, but are not limited to: gelatin,carboxymethylcellulose, butylhydroxytoluene, chitosan, etc.

In certain embodiments of the invention, tackifying agents are used tomodify adhesion to the biological substrate while simultaneouslycreating a procoagulant. In certain embodiments, the tacking agents arepresent in concentrations of from about 0.1 to about 10% (w/w), such asfrom about 0.1 to about 4% (w/w).

Optional Components

The above described substrate component of the subject compositions may,in certain embodiments, include one or more optional components thatmodify the properties of the phase invertible composition produced fromthe substrate and crosslinker. Representative optional components ofinterest are now discussed in greater detail below.

Plasticizing Agents

In accordance to the invention, a plasticizing agent may be present inthe substrate. The plasticizing agent provides a number of functions,including wetting of a surface, or alternately, increasing the elasticmodulus of the material, or further still, aiding in the mixing andapplication of the material. Numerous plasticizing agents exist,including fatty acids, e.g., oleic acid, palmitic acid, etc.,dioctylphtalate, phospholipids, and phosphatidic acid. Becauseplasticizers are typically water insoluble organic substances and arenot readily miscible with water, it is sometimes advantageous to modifytheir miscibility with water, by pre-mixing the appropriate plasticizerwith an alcohol to reduce the surface tension associated with thesolution. To this end, any alcohol may be used. In one representativeembodiment of this invention, oleic acid is mixed with ethanol to form a50% (w/w) solution and this solution then is used to plasticize theproteinaceous substrate during the formulation process. Whereas the typeand concentration of the plasticizing agent is dependent upon theapplication, in certain embodiments the final concentration of theplasticizing agent is from about 0.01 to 10% (w/w), including from about2 to about 4% (w/w). Other plasticizing agents of interest include, butare not limited to: polyethylene glycol, glycerine, butylhydroxytoluene,etc.

Carbohydrate Procoagulant

In certain embodiments, the substrates include a carbohydrateprocoagulant. Chitosan and derivates of chitosan are potent coagulatorsof blood and, therefore, are beneficial in formulating sealant materialscapable of sealing vascular injuries. While virtually all chitinmaterials have been demonstrated to have some procoagulant activity, inaccordance to the invention, the use of acetylated chitin is employed asan additive for the formulation of sealant intended for blood control.Acetylation of the molecule can be achieved in a number of differentways, but one common method is the treatment of chitosan/acetic acidmixtures with acid anhydrides, such as succinic. This reaction isreadily carried out at room temperature. In accordance with theinvention, gels created in this manner combined with proteinaceoussubstrates and crosslinked in situ are beneficial for the creation of abiocomposite structural member. As such, the carbohydrate procoagulantmay be chitosan, low molecular weight chitosan, chitin, chitosanoligosaccharides, and chitosan derivatives thereof. In accordance withthe teachings of this invention the carbohydrate component, e.g.,chitosan, may be present in concentrations ranging from about 0 to about20%, such as from about 0.1 to about 5% (w/w).

Fillers

Fillers of interest include both reinforcing and non-reinforcingfillers. Reinforcing fillers may be included, such as chopped fibroussilk, polyester, PTFE, NYLON, carbon fibers, polypropylene,polyurethane, glass, etc. Fibers can be modified, e.g., as describedabove for the other components, as desired, e.g., to increasewettability, mixability, etc. Reinforcing fillers may be present fromabout 0 to 40%, such as from about 10 to about 30%. Non-reinforcingfillers may also be included, e.g., clay, mica, hydroxyapatite, calciumsulfate, bone chips, etc. Where desired, these fillers may also bemodified, e.g., as described above. Non-reinforcing fillers may bepresent from about 0 to 40%, such as from about 10 to about 30%.

Biologically Active Agents

Biologically active agents may be included, e.g., bone growth factors,tissue activators, cartilage growth activators, small molecule activeagents, etc. Thus, the biologically active agents can include peptides,polypeptides, proteins, saccharides, polysaccharides and carbohydrates,nucleic acids, and small molecule organic and inorganic materials.Specific biologically active agents include antibiotics, antivirals,steroidal and non-steroidal anti-inflammatories, antineoplastics,anti-spasmodics including channel blockers, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, enzymes and enzyme inhibitors,anticoagulants, growth factors, DNA, RNA and protein synthesisinhibitors, anti-cell migratory agents, vasodilators, and other drugsused for treatment of injury to tissue. Examples of these compoundsinclude angiotensin converting enzyme inhibitors, anti-thromboticagents, prostacyclin, heparin, salicylates, thrombocytic agents,anti-proliferative agents, nitrates, calcium channel blocking drugs,streptokinase, urokinase, tissue plasminogen activator (TPA) andanisoylated plasminogen activator (PA) and anisoylatedplasminogen-streptokinase activator complex (APSAC), colchicine andalkylating agents, growth modulating factors such as interleukins,transformation growth factor P and congeners of platelet derived growthfactor, monoclonal antibodies directed against growth factors, modifiedextracellular matrix components or their receptors, lipid andcholesterol sequestrants and other agents which may modulate vesseltone, function, arteriosclerosis, and the healing response to vessel ororgan injury post intervention.

Foaming Agent

In certain embodiments, the substrate may include a foaming agent which,upon combination with the crosslinker composition, results in a foamingcomposition, e.g., a composition that includes gaseous airbubblesinterspersed about. Any convenient foaming agent may be present, wherethe foaming agent may be an agent that, upon contact with thecrosslinking composition, produces a gas that provides bubble generationand, hence, the desired foaming characteristics of the composition. Forexample, a salt such as sodium bicarbonate in an amount ranging fromabout 2 to about 5% w/w may be present in the substrate. Uponcombination of the substrate with an acidic crosslinker composition,e.g., having a pH of about 5, a foaming composition is produced.

Additional Modifiers

Additional modifiers may also be present. For example, blends of one ormore polymers (e.g., polyblends), such as Teflon, PET, NYLON, hydrogels,polypropylene, etc., may be present. The polyblends may be modified,e.g., as described above, to provide for desired properties. Theseadditional modifiers may be present in amounts ranging from about 0 to50%, including from about 10 to about 30%.

Buffer

Upon mixture of the proteinaceous substrate and crosslinker to producethe subject phase invertible composition, buffering of the phaseinvertible composition is employed in certain embodiments for a numberof reasons, e.g., to optimize the bonding strength of the composition tothe attaching surface, to optimize the conditions necessary for internalcrosslinking to occur, etc. For example, optimum crosslinking forproteins using glutaraldehyde crosslinkers occurs at pH range from about6 to about 8. Buffers capable of maintaining this range are useful inthis invention, as long as they do not interfere with the carbonylterminal of the crosslinker or modify the amine terminus of the aminoacids. For example, phosphate buffers have a pKa value in the range ofpH 7.0 and do not interfere with the crosslinking process because theydo not contain carboxylic or amine functionalities. Phosphate buffer upto 1 M in strength is suitable for use as a buffer in the presentinvention, where in certain embodiments the phosphate buffer is about0.01 to about 0.3M in strength. While phosphate buffering of thesolutions is ideal for the stability of the protein substrate inapplications where increased adhesion is required, an acidic buffer maybe used as well. Citrate buffers 0.1-1 M and having a pH range of about4.5 to about 6.5 have been found to be useful for this invention.

The buffer may be present in either the initial crosslinker component orthe initial proteinaceous substrate component, or present in bothcomponents, as desired.

Combination of Substrate and Crosslinker to Produce Phase InvertibleComposition

As summarized above, the subject phase invertible compositions areprepared by combining a liquid proteinaceous substrate and a liquidcrosslinker in appropriate amounts and under conditions sufficient forthe phase invertible composition to be produced. In certain embodiments,the substrate and crosslinker are combined in a ratio (v/v) ranging fromabout 1/5 to about 5/1; so that a resultant phase invertible compositionis produced in which the total protein concentration ranges from about10 to about 60%, such as from about 20 to about 50%, including fromabout 30 to about 40% and the total crosslinker composition ranges fromabout 0.1 to about 20%, such as from about 0.5 to about 15%, includingfrom about 1 to about 10%.

Combination of the substrate and crosslinker typically occurs undermixing conditions, such that the two liquid components are thoroughlycombined or mixed with each other. Combination or mixing may be carriedout using any convenient protocol, e.g., by manually combining twocomponents, by employing a device that combines the two components, etc.Combination or mixing is typically carried out at a temperature rangingfrom about 20 to about 40.degree. C., such as room temperature.

Combination of the proteinaceous substrate and crosslinker as describedabove results in the production of a phase invertible composition. Byphase invertible composition is meant a composition that goes from afirst liquid state to a second non-fluid, e.g., gel or solid, state. Inthe second non-fluid state, the composition is substantially, if notcompletely, incapable of fluid flow. The phase invertible compositiontypically remains in a fluid state, following combination of thesubstrate and crosslinker components, for a period of time ranging fromabout 10 seconds to about 10 minutes, such as from about 20 seconds toabout 5 minutes, including from about 30 seconds to about 120 second,when maintained at a temperature ranging from about 15.degree. C. toabout 40.degree. C., such as from about 20.degree. C. to about30.degree. C.

Specific phase invertible formulations include as components: (i) afirst composition comprising a proteinaceous substrate compositionhaving about 30%-50% albumin, and about 0.1%-0.3% chitosan chloride; and(ii) a second composition comprising a crosslinking composition havingabout 3%-10% heat treated glutaraldehyde, about 0.1%-1% hyaluronic acid,and optionally, about 0%-1.5% sodium salt of carboxymethylcellulose highviscosity. Such phase invertible formulations generally have an averagecure time of about 5-60 seconds upon admixing of the first and secondcompositions, and an average burst of about 125-1000 mmHg, and moretypically, an average cure time of about 10-30 seconds, and an averageburst of about 200-850 mmHg, as measured at about room temperature.

In a particular embodiment, a phase invertible formulation is providedthat includes as components: (i) a first composition comprising aproteinaceous substrate composition having about 40% albumin, and about0.2% chitosan chloride; and (ii) a second composition comprising acrosslinking composition having about 4.4%-7.5% heat treatedglutaraldehyde, about 0.2% hyaluronic acid, and optionally, about0%-0.75% sodium salt of carboxymethylcellulose high viscosity. Suchphase invertible formulations generally have an average cure time ofabout 10-30 seconds upon admixing of the first and second compositions,and an average burst of about 250-800 mmHg, and more typically, anaverage cure time of about 10-25 seconds, and an average burst of about350-700 mmHg, as measured at about room temperature.

As also described above, various additional materials can beincorporated into the phase invertible compositions to serve any ofseveral purposes, such as to modify the physical characteristics of thecomposition, and/or aid in the repair of a target tissue or biologicalmaterial to which the composition is applied. For example, biologicallyactive agents such as peptides, proteins, nucleic acids, carbohydratemolecules, small molecules and the like can be incorporated to attractand bind specific cell types, such as white cells and platelets, ormaterials such as fibronectin, vimentin, and collagen, can be used toenhance healing by non-specific binding. Tracing material, such asbarium, iodine or tantalum salts, may also be included to allowvisualization and/or monitoring of the phase invertible composition. Incertain embodiments, different biologically active agents can be used indifferent applications or layers when one or more phase invertiblecompositions is differentially applied.

In certain embodiments, cells can also be incorporated into or appliedin connection with the phase invertible compositions before, duringand/or after application of the composition. When employed, cells aregenerally included in the proteinaceous substrate composition, orapplied in conjunction with or separately from the phase invertiblecomposition in a manner that allows their adherence at the site ofapplication. The cells can be living, artificial cells, cell ghosts(i.e., red blood cell or platelet ghosts), or pseudovirions, dependingon a given end use. For example, the cells may be selected to producespecific agents such as growth factors at the site of application.Biologically active agents that modulate cell viability,differentiation, and/or growth can be included as well. In someembodiments, the cells may be stem cells, or in other embodiments,progenitor cells corresponding to the type of tissue at the treatmentlocation or other cells, providing therapeutic advantages. For example,liver cells incorporated into the phase invertible composition andapplied to a wound or surface of the liver of a patient may aid in itsregeneration and repair. This may be particular useful in cases wherediseases such as cirrhosis, fibrosis, cystic disease or malignancyresults in non-functional tissue, scar formation or tissue replacementwith cancerous cells. Similar methods may be applied to other organs andtissues as well.

The biologically active agents can be incorporated physically and/or bychemical attachment. Physical incorporation is carried out by mixing thebiologically active agent with the phase invertible material prior toand/or during application to the target surface and curing. The materialis usually mixed into the proteinaceous substrate solution to form asolution, suspension or dispersion. In one embodiment, the biologicallyactive agent can be encapsulated within delivery devices such asmicrospheres, microcapsules, liposomes, cell ghosts or pseudovirions,which in themselves effect release rates and uptake by cells. Chemicalincorporation of the biologically active agent is carried out bychemically coupling the agent to a polymeric material of either theproteinaceous substrate or crosslinking composition, usually theproteinaceous substrate, before or at the time of polymerization (e.g.,by conjugation through reactive functional groups, such as amines,hydroxyls, thiols, and the like). To ensure that the desired cure timesand burst properties of the phase invertible composition are maintained,physical and/or chemical incorporation of a biologically active agenttakes into consideration the relative amounts of proteinaceous substrateand crosslinker present in the final composition, and can be adjustedaccordingly.

Methods

The subject biocompatible phase invertible compositions are employed inmethods where a quantity of the phase invertible composition isdelivered to a particular site or location of a subject, patient or hostin need thereof. The subject, patient or host is typically a “mammal” or“mammalian,” where these terms are used broadly to describe organismswhich are within the class mammalian, including, but not limited to, theorders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guineapigs, and rats), lagomorpha (e.g. rabbits) and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the animals or hosts,i.e., subjects (also referred to herein as patients) will be humans.

The quantity that is delivered to the subject in any given applicationwill necessarily vary depending on the nature of the application and useof the composition, but in certain representative embodiments rangesfrom about 1 to about 50 ml, such as from about 1 to about 25 ml,including from about 1 to about 5 ml, e.g., about 3 ml.

While necessarily dependent on the particular application in which thesubject composition is being employed, the subject composition is, inmany embodiments, locally delivered to a particular region, site orlocation of the host, where the site or location may, of course, vary.Representative sites or locations include, but are not limited to:vessels, organs, and the like. Depending on the particular application,the composition may be delivered to the site of interest manually orwith a delivery device, e.g., the delivery device employed to deliverthe composition in stenting applications, described in greater detailbelow.

Utility

The subject biocompatible phase invertible compositions find use in avariety of different applications. Representative applications of thesubject phase invertible compositions include those described in U.S.Pat. Nos. 3,438,374; 5,092,841; 5,292,362; 5,385,606; 5,575,815;5,583,114; 5,843,156; 6,162,241; 6,290,729; 6,302,898; 6,310,036;6,329,337; 6,371,975; 6,372,229; 6,423,333; 6,458,147; 6,475,182;6,547,806; and 7,303,757; as well as U.S. Application Nos. 2002/0015724;2002/0022588; 2002/0133193; 2002/0173770; 2002/0183244; 2002/019490;2002/0032143; the disclosures of which are herein incorporated byreference.

Systems

Also provided are systems for use in practicing the subject methods. Thesystems may include fluid delivery elements for delivery of thesubstrate and crosslinking composition to the site of administration,mixing elements, etc. Examples of such systems include those describedin U.S. Pat. No. 7,303,757; the disclosure of which is hereinincorporated by reference.

Kits

Also provided are kits for use in practicing the subject methods, wherethe kits typically include a distinct liquid substrate and liquidcrosslinking composition components of a phase invertible fluidcomposition, as described above. The substrate and crosslinkingcompositions may be present in separate containers in the kit, e.g.,where the substrate is present in a first container and the crosslinkingagent is present in a second container, where the containers may or maynot be present in a combined configuration.

The subject kits may also include a mixing device, for mixing thesubstrate and crosslinker together to produce the phase invertiblecomposition. The kits may also include a delivery device (which may ormay not include a mixing element), such as a dual barrel syringe,catheter devices, and the like, as described above.

The kit may further include other components, e.g., guidewires, sensorwires, etc., which may find use in practicing the subject methods.

In addition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

The following examples are provided by way of illustration and not byway of limitation.

EXPERIMENTAL

Various formulations of phase invertible compositions were prepared inwhich the sodium salt of hyaluronic acid (HA) was included in theprotein component and the crosslinking component. Illustrativeformulations were tested as shown in Table 1.

TABLE-US-00001 TABLE 1 Protein Formulation Crosslink Formulation Lot#Alb HA Chit C1 213 HTGA HA CMC High V is 004-49 40% 0.0% 0.2% 7.5% 0.2%0.00% 004-42-1 40% 0.2% 0.2% 4.4% 0.0% 0.75% 004-42-2 40% 0.2% 0.2% 7.5%0.0% 0.75% 004-42-3 40% 0.2% 0.2% 4.4% 0.0% 0.00% 003-118-2 40% 0.0%0.2% 7.5% 0.2% 0.75% Abbreviations: Alb=Albumin; HA=Hyaluronic acid;Chit C1 213=Chitosan Chloride; HTGA=heat treated glutaraldehyde; and CMCHigh Vis=the sodium salt of carboxymethylcellulose high viscosity.

The protein solution is prepared in a 10 mM, pH 6.2 phosphate buffersolution. The cross-linker solution is prepared in a 10 mM, pH 7phosphate buffer solution.

An in-vitro test system was used to determine the burst strength of eachformulation. A one inch square piece of porcine aorta wall is fastenedto a pressure vessel over a 5 mm diameter hole. A hole is puncturedthrough the aorta using a 14 gauge needle. The sealant is applied to theaorta over the 14 ga needle hole and allowed to cure. Air pressure isapplied to the inside of the pressure vessel until the sealant ruptures.The air pressure required to cause rupture is measured using amanometer.

The observed properties of these formulations are shown in Tabular formin FIG. 1, and in Table 2, for the aorta test system.

TABLE-US-00002 TABLE 2 Average Cure Lot# Time (sec) n=2 Average Burst(mmHg) n=2 004-49 19.50 565.20 004-42-1 22.00 353.70 004-42-2 13.50566.20 004-42-3 33.50 246.20 003-118-2 11.50 499.20

The results in FIG. 1 and Table 2 demonstrate that when hyaluronic acid(HA) is provided in the crosslinking component and not the proteincomponent, undesirable interactions with the protein component areeliminated and the viscosity of the crosslinking component is enhanced,which results in an improved overall 2 component sealant composition.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A method of producing a phase invertiblecomposition, said method comprising: combining: (a) a liquidproteinaceous substrate; and (b) a liquid crosslinking compositioncomprising a macromolecular crosslinking agent which comprises areaction product of an excess of a liquid aldehyde crosslinking agentand a glycosaminoglycan, wherein the liquid crosslinking composition hasbeen formed prior to combining with the liquid proteinaceous substrate;to produce said phase invertible composition.
 2. The method according toclaim 1, wherein said glycosaminoglycan is hyaluronan.
 3. The methodaccording to claim 1, wherein said proteinaceous substrate comprises aproteinaceous material selected from the group consisting of: albumin,elastin, fibrin and soluble and insoluble forms of collagen andcombinations thereof.
 4. The method according to claim 3, wherein saidcrosslinking agent comprises glutaraldehyde.
 5. The method according toclaim 4, wherein said glutaraldehyde is heat stabilized.